Isolation and Characterization of a Putative Keratin-Associated Protein Gene Expressed in Embryonic Skin of Mice Mikiro Takaishi, Yoshimi Takata, Toshio.

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Isolation and Characterization of a Putative Keratin-Associated Protein Gene Expressed in Embryonic Skin of Mice Mikiro Takaishi, Yoshimi Takata, Toshio Kuroki, Nam-ho Huh Journal of Investigative Dermatology Volume 111, Issue 1, Pages 128-132 (July 1998) DOI: 10.1046/j.1523-1747.1998.00241.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification of differentially expressed gene fragments by RNA differential display. Total RNA samples from embryonic mouse epidermis at 12.5 dpc (lane 1), 13.5 dpc (lane 2), 14.5 dpc (lane 3), 15.5 dpc (lane 4), and 16.5 dpc (lane 5) were reverse-transcribed and amplified by PCR with a primer set of 5′-TTTTTTTTTTTTMC-3′ and 5′-GGTACTCCAC-3′. The PCR products were analyzed on 6% polyacrylamide gel electrophoresis. Small circles are the marks for recovering DNA fragments. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 4C32 cDNA encodes a protein with characteristic amino acid repeats. (a) Nucleotide sequence of 4C32 is numbered from the 5′-end of the three longest cDNA clones isolated independently. The putative initiation codon is shown in bold, the six 10 peptide repeats are underlined, and the polyadenylation signal is boxed. This nucleotide sequence was registered in GenBank under the accession number of AF031485. (b) Repetitive amino acid sequence of 4C32. CQ motif (in bold) appears in every 10 amino acids. The amino acid sequence is partially conserved among the repetitive units. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 4C32 is expressed in the uppermost layer of developing mouse skin. Total RNA was prepared from the epidermis (lanes 1–4) and the dermis (lanes 5–8) of embryonic ICR mice at 13.5 dpc (lanes 1 and 5), 14.5 dpc (lanes 2 and 6), 15.5 dpc (lanes 3 and 7), and 16.5 dpc (lanes 4 and 8). The probes used were 4C32 (a), loricrin (b), cytokeratin K10 (c), cytokeratin K14 (d), and glyceraldehyde-3-phosphate dehydrogenase (e) as a control. 28S and 18S (a) designate the position of corresponding ribosomal RNA. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 4C32 transcripts in skin are undetectable from 17.5 dpc onwards. RNA was prepared from mouse skin at the age of 16.5 dpc (lane 1), 17.5 dpc (lane 2), 18.5 dpc (lane 3), 1 d (lane 4), 3 d (lane 5), and 1 wk (lane 6). The probes used were 4C32, loricrin (Lor), and glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Detection of 4C32 transcripts in dorsal skin, tongue, and tail by in situ hybridization. Digoxigenin-labeled anti-sense RNA probe of 4C32 was hybridized to the dorsal skin (a) and the tongue (b) of 16.5 dpc embryo, and the tongue (c) and the tail (d) of young adult mouse. In (c) and (d) the anterior is to the right. Scale bars: (a, b) 50 μm; (c, d) 100 μm. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 4C32 is expressed in skin, but not in oral mucosa. Serial sections were made from the lower lip of 16.5 dpc ICR mouse embryo, and stained with hematoxylin and eosin (a) or hybridized with anti-sense RNA probe of 4C32 (b), loricrin (c), cytokeratin K10 (d), K14 (e), and K13 (f). Hybridization with corresponding sense probes was performed in parallel, giving consistently negative signals. Scale bar, 100 μm. Journal of Investigative Dermatology 1998 111, 128-132DOI: (10.1046/j.1523-1747.1998.00241.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions